Mucins are a family of large and heavily O-glycosylated glycoproteins synthesized by all wet-surfaced epithelia, including the corneal and conjunctival epithelia. The ocular surface epithelia produce the secreted mucin MUC5AC and the membrane-associated mucins MUC's 1, 4 and 16. Terminal carbohydrates on mucins are the most exposed to the extracellular milieu, but little is known about their roles and character in each mucin at the ocular surface. Our previous studies have demonstrated that binding of an antibody specific to a terminal carbohydrate on MUC16 is altered in dry eye patients. This proposal is aimed at identifying terminal carbohydrate structures on individual ocular mucins, the enzymes involved in their biosynthesis and the role of terminal O-glycans in maintaining a wet ocular surface and preventing pathogen invasion. AIM I: We hypothesize that individual mucins are differentially O-glycosylated by the ocular surface epithelia. We propose to: A. Characterize the repertoire of terminal O-linked carbohydrates on individual mucins isolated from tears of normal individuals. B. Identify and localize glycosyltransferases (sialyltransferases) responsible for their biosynthesis. C. Test in vitro the role of terminal O-linked carbohydrates on mucins of the glycocalyx in conferring disadhesive properties to epithelial cells and resistance to transfection with adeno-associated viruses. AIM II: We hypothesize that in dry eye patients there is an alteration in terminal O-glycan structures of the membrane-associated mucins that affects hydration of the ocular surface. We propose to: A. Determine if there is an alteration of terminal carbohydrates of each membrane-associated mucin purified from the apical tear surface of dry eye patients. B. Determine if there is an alteration in the expression of glycosyltransferases (sialyltransferases) in dry eye patients. C. Determine in vitro whether the depletion of terminal carbohydrates on mucins in corneal and conjunctival cells enhances the penetrance of rose bengal into these cells. AIM III: We hypothesize that O-linked carbohydrates present on the secreted mucin MUC5AC in the tear fluid have specific affinity to P. aeruginosa, preventing their attachment to the ocular surface glycocalyx and facilitating their clearance. We propose to: A. Compare the binding of P. aeruginosa to the O-linked carbohydrates of individual mucins collected from tears. B. Determine in vitro how inhibition of O-glycan synthesis on membrane-associated mucins affects P. aeruginosa adherence to corneal cells. [unreadable] [unreadable]